Synaptic transmission in the brain is initiated by neurotransmitter release, which is mediated by synaptic vesicle exocytosis. Insights into the mechanisms of neurotransmitter release and its regulation are essential for understanding how the brain processes information, and how synaptic transmission is affected in diseases such as Parkinson's disease, Alzheimer's disease, and drug addiction. Release is governed by a complex protein machinery that includes as central components proteins with homologues in most types of intracellular membrane fusion such as the SNARE proteins and munc18-1. In addition, several proteins such as munc13-1 and complexins play critical roles that are specialized for the tight spatial and temporal regulatory requirements of neurotransmitter release. While the research performed under this grant and studies from other laboratories have yielded key insights into how these proteins function, the mechanism of release is still unclear and fundamental questions remain unanswered. The research proposed in this application is designed to build on this success and on crucial findings made in preliminary experiments that suggest novel interactions between these key components and new hypotheses on how they function. These include an interaction between munc18-1 and the SNAREs that suggest a novel model for the core of the fusion machinery, and a munc18-1/munc13-1 interaction that suggest a novel mechanism for the priming step that leaves synaptic vesicles ready for neurotransmitter release. This research involves an interdisciplinary approach integrating structural studies at atomic resolution, biochemical experiments, reconstitution assays and electrophysiological analyses of neurotransmitter release in neurons. Four specific aims are proposed that focus on: 1. Munc 18-1/SNARE interactions;2. Mechanisms of munc13-1 action and its regulation;3. Complexin/SNARE interactions;and 4. Reconstitution of basic steps of neurotransmitter release. The results of this research will not only be important to understand the mechanisms of neurotransmitter release and membrane fusion in general, but will also have an impact in the design of therapies for brain disorders that involve changes in synaptic transmission.